NAD(P)H:quinone oxidoreductase (NQO1, EC 1.699.2, DT-diaphorase) is a ubiquitous enzyme that carries out 2-electron reduction of numerous quinone substrates and is induced in antioxidant responses. Although a "physiologic" substrate for NQO1 had been obscure, we observed recently that NQO1 reduces the endogenous alpha-tocopherol (alphaTH, vitamin E) oxidation product alpha- tocopherolquinone (TQ) to alpha-tocopherolhydroquinone (TQH2), which can, in turn, regenerate alphaTH from the alpha- tocopheroxyl radical. An alphTH regenerating system is thought to be an essential contributor to cellular antioxidant protection. We hypothesize that an essential function of NQO1 is to supply reducing equivalents to redox cycles of alphaTh, thus sustaining cellular antioxidant protection against propagation of oxidative damage in membranes. This action may involve either reduction of one-electron oxidized tocopheroxyl radicals or 2- electron oxidized tocopherones either by NQO1 itself or by TQH2. To test these hypotheses, we propose: Aim 1) To characterize the participation of TQH2 and NQO1 in a one-electron alphaTH redox cycle. Reduction of the tocopheroxyl radical to alphaTH either directly by NQO1 or via TQH2 will be studied in liposome and membrane systems. Aim 2) To characterize the participation of TQH2 and NQO1 in a two-electron alphaTh redox cycle. Reduction of 8a-substituted tocopherones to alphaTH either directly or via TQH2 will be studied in liposome and membrane models. Aim 3) To characterize the impact of NQO1 on alphaTh redox status and antioxidant turnover in intact cells. These studies will employ CHO cells and human cell lines expressing NQO1 polymorphisms. The levels and distribution of alphaTH and its oxidation products will be measured as a function of NQO1 activity in cells subjected to oxidative stress. These studies will clarify the endogenous antioxidant role of NQO1 and determine how the antioxidant chemistry of alphaTH, Nature's most ubiquitous membrane antioxidant, is tied to cellular metabolic redox balance. Answers to these questions are essential to improving our understanding of the interplay between cellular antioxidant defenses and their roles in human health and disease.